1. 12 August 2003 AmpC  -Lactamases & their detection David Livermore Health Protection Agency, Colindale, London

2.  -Lactamase-stable cephs CONH O COOH R C N OR H2NH2N Oxyimino-aminothiazolyl stability to classical TEM/SHV SN S N

3.  -Lactamase classes

4. AmpC enzymes Mol wt. c. 40,000; alkaline pI Hydrolytic activity 1 st gen cephs –rapid 2/3 ceph cephs –slow but kinetically efficient 4-gen cephs –slow, kinetically inefficient Carbapenems –nearly stable…. not quite! Not inhibited by clavulanate; poorly inhibited by sulphones

5. AmpC  -lactamases Basal in: E. coli & shigellae Inducible in: Enterobacter spp. C. freundii M. morganii Serratia spp. P. aeruginosa 2nd, 3rd gen cephs: Labile, but weak inducers, select derepressed mutants [  -lactam] Amt  -lactamase Derepressed Inducible

6. Induction of AmpC Cell wall constantly re-cycled Yields disaccharide tri-peptides (DTPs) –Absorbed by AmpG –Cleaved by AmpD = N-acetyl-muramyl-L-alanine amidase Excess DTPs bind AmpR, activating ampC

7. Uninduced AmpC Wall fragments recycled by AmpD AmpR in repressor conformation ampC (  -lactamase gene) NOT expressed ampCamp R ampD AmpDAmpR

8. Induced AmpC ampCamp R ampD More recycling: AmpD overwhelmed Wall fragments convert AmpR to activator ampC (  -lactamase gene) expressed AmpD  -lactamase

9. Derepressed AmpC ampCamp R ampD ampD inactivated by mutation AmpR constantly converted to activator ampC hyper-expressed  -lactamase++

10. Strong & weak inducers Strong inducerWeak inducer Labile1 st gen cephs Ampicillin 3 rd gen cephs Piperacillin Aztreonam StableImipenem(Temocillin) (Meropenem) Strong inducer induces below MIC, weak doesn’t

11. MICs (mg/L) for E. cloacae AmpC mutants

12. MICs (mg/L) for P. aeruginosa AmpC mutants

13. Strong inducers –e.g. imipenem What selects derepression? No! – derepressed no more R than inducible All weak inducers? No- Not if they are stable LABILE weak inducers? Yes! Derepressed are R, whilst inducible cells are S, so derepressed are selected

14. Over-run by mutants Derepression occurs in 1 cell / 10 7 Confers resistance to 3-gen cephs Overnight, one cell can give 10 9 progeny Selection in therapy can cause R x failure... Mutant emerges randomly Sensitive cells killed by antibiotic Mutant’s progeny overrun

15. Initial isolation of 3 rd -gen cephR Enterobacter, & prior R x Chow et al. Ann Intern Med 1991, 115, 585-90

16. Selecting Enterobacter R to 3 rd gen cephs during R x Kaye et al. AAC 2001, 45, 2628; Cosgrove et al. Arch Intern Med 2002, 162, 185

17. Plasmidic AmpC enzymes Escaping from enterobacterial and Aeromonas chromosomes Many good reports since 1991 CMY, MOX, FOX, LAT, DHA, ACT & BIL enzymes

18. Sources of plasmid AmpC ClassSourceExamples CITC. freundiiCMY-2 to 7; LAT-1,3,4 ENTEnterobacter spp.ACT-1; MIR-1 FOXAeromonas spp.FOX-1 to -5; MOXAeromonas spp.MOX-1,-2; CMY-1,7 & 8 DHAM morganiiDHA-1, -2 ACCH. alveiACC-1

19. Plasmid AmpC Jenks et al. 1995. J Antimicrob Chemother 35, 235-6.

20. Enzymes in 1127 cephR isolates from 16 labs in S England, 2004 Potz et al., JAC, 2006 in press

21. Prevalence of mechanisms: BSAC bacteraemia surveillance, 2005 http://www.bsacsurv.org

22. Inducible AmpC Cefoxitin Ceftazidime But mutational derepression is the problem, not induction Better predict risk from species I/D

23. Suspect derepressed / plasmid AmpC if: Resistant 3-gen cephs, NOT cefepime & cefpirome Resistant to cefoxitin (but more ESBL producers R, too, nowadays) No ceph/clav synergy

24. Geometric mean MICs (mg/L): AmpC producers; 2004 London SE survey E. coli AmpC E. coli ESBL Enterobacter AmpC Enterobacter ESBL Cefotaxime122287249 Ceftazidime18393681 Cefepime0.38390.914.4 Cefpirome0.57532.410 Cefuroxime35>64 Cefoxitin5117>6451

25. Some wrinkles… AmpC-derepressed M. morganii are S to pip/tazo AmpC derepressed Serratia are S to ceftazidime Cefoxitin R an unreliable marker for Providencia, Morganella & Serratia spp. –Inducible & derepressed strains may appear I or S AmpC derepressed P. aeruginosa tend to be S to carbenicillin / efflux mutants are R Life complicated if there’s an ESBL with the AmpC

26. Confirmatory tests for AmpC Seek cefotaxime/cloxacillin synergy –Cefotaxime MIC +100 mg/l cloxacillin –Zones of cefotaxime 30  g discs on agar + 100 mg/L cloxacillin –No agreed interpretive standards Can also use phenylboronic acid as inhibitor DHA enzymes inducible & especially difficult to detect

27. Cefotaxime combinations vs. AmpC E. coli: London SE survey

29. Cefotaxime / cloxacillin tests for AmpC

30. 3-D test for AmpCs Plate seeded with cefoxitin S indicator strain Cut cross in agar, heavily inoculated with test strain Cefoxitin disc Looks for distortion where cross intersects the cefoxitin zone

31. Clover leaf (3 dimensional) test for AmpC Test strain E. cloacae, AmpC derepressed Indicator E. coli NCTC10418 Disc Cefoxitin 30  g

32. Clover leaf (3 dimensional) test for cephalosporinase Test strain E. cloacae, AmpC derepressed Indicator E. coli NCTC10418 Disc Cefotaxime 30  g

33. Phenyl boronic acid for detection of plasmid AmpC Expansion (mm) of FOX 30 zone with 400  g phenyl boronic acid Fold MIC reduction for cefoxitin + 400 mg/L phenyl boronic acid Kleb MOX-112128 E. coli LAT-21264 Kleb DHA-114128 Kleb DHA-21364 E. coli ACC-144 Kleb ACT-11364 ALL ESBL +ve<2<2<2<2 Coudron JCM 2005 43 4163

34. Disc tests for AmpC 60 E. coli & Klebsiella : cefoxitin MICs reduced >4-fold by 100 mg/L cloxacillin % with >5 mm zone expansion Cefoxitin + cloxacillin 100  g 86% Cefoxitin + BZB 64  g 89% Cefpodoxime + BZB 64  g 97% Cefpodoxime + clav + BZB 64  g 100% BZB: benzo(b)thiophene-2-boronic acid Brenwald et al., JAC 2005, 56, 600

35. Cefoxitin R isolates in phenyl boronic acid / cefoxitin tests 3-DPhenyl boronic, disc Phenyl boronic MIC +-+-+- K. pneumoniae106161101211012 K. oxytoca425151 E. coli229340753976 P. mirabilis244253262 Coudron JCM 2005 43 4163

36. Multiplex detection of plasmid AmpC genes Method of Perez-Perez & Hanson JCM 2002, 40, 2153

37. AmpC hyperproducers : options Active Carbapenems Temocillin 4-gen cephs Not active Penicillins except temocillin Inhibitor combinations 1, 2, 3-gen cephs Aztreonam

38. AmpC Summary Mutant selection the problem, not induction Selection mostly in therapy with 3-gen cephs AmpC types spreading to plasmids Suspect AmpC if: –R 3 rd, not 4 th gen cephs; cefoxitin R, no ceph/clav synergy Confirmatory tests poorly standardised, exploit synergy with cloxacillin or phenyl boronic acids